EP3813791A2 - Stabile lyophilisierte darreichungsform von protein - Google Patents

Stabile lyophilisierte darreichungsform von protein

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Publication number
EP3813791A2
EP3813791A2 EP19759046.6A EP19759046A EP3813791A2 EP 3813791 A2 EP3813791 A2 EP 3813791A2 EP 19759046 A EP19759046 A EP 19759046A EP 3813791 A2 EP3813791 A2 EP 3813791A2
Authority
EP
European Patent Office
Prior art keywords
protein
dosage form
formulation
lyophilized dosage
stable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19759046.6A
Other languages
English (en)
French (fr)
Inventor
Prasanna DEVARANENI
Vaibhav DEOKAR
Rustom Mody
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lupin Ltd
Original Assignee
Lupin Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lupin Ltd filed Critical Lupin Ltd
Publication of EP3813791A2 publication Critical patent/EP3813791A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • the present invention relates to a stable lyophilized dosage form of protein pharmaceuticals. Specifically the present invention relates to a stable lyophilized dosage form of protein which upon reconstitution has the same composition as that of an already established or developed ready-to-use liquid dosage form of the same protein.
  • ready-to-use liquid and lyophilized formulation remain the primary dosage forms. It is difficult to have the same composition for ready-to-use liquid formulation and lyophilized dosage forms due to uncertainty during development, most importantly due to different stability requirements of dmg molecule in liquid and in lyophilized formulations. From a commercial point of view, direct conversion of already established or developed ready-to-use liquid composition of protein into a lyophilized dosage form may pose additional challenges for development due to specific attributes of lyophilized dosage form such as 1) elegant & stable cake, which is free from collapse or melt back 2) minimum reconstitution time 3) minimum particulates upon reconstitution 4) long term stability. Similarly conversion of already established or developed lyophilized dosage form into a ready-to-use liquid formulation, both having the same composition, also has its own challenges.
  • a stable lyophilized dosage form of protein which upon re-constitution has the same composition as that of ready-to-use liquid dosage form of the same protein, containing commercially desired attributes, can be prepared if the excipients of ready-to-use liquid formulation are divided in two portions of lyophilized formulation i.e. lyophilate and reconstitution liquid.
  • the invention is related to a stable lyophilized dosage form of protein which upon re-constitution is qualitatively as well as quantitatively same as that of ready- to-use liquid dosage form of the same protein.
  • the invention relates to a stable lyophilized dosage form of protein which upon re-constitution is qualitatively as well as quantitatively same as that of ready-to-use liquid dosage form of the same protein wherein the lyophilized dosage form comprises of a lyophilate and a reconstitution liquid.
  • a stable lyophilized dosage form of protein comprising a lyophilate and a reconstitution liquid, forms a solution upon reconstitution which is qualitatively as well as quantitatively same as that of ready-to-use liquid dosage form of the same protein.
  • the invention is related to a stable lyophilized dosage form of a protein comprising a lyophilate with reduced concentration of salt.
  • a stabilized lyophilized formulation of a protein having an elegant cake can be prepared by limiting the salt concentration from the lyophilate during lyophilization process.
  • a stable lyophilized dosage form of protein comprising a lyophilate with a reduced concentration of salt as well as buffer.
  • elegant cake formation can be prepared by controlling the ionic strength of lyophilate.
  • a lyophilized dosage form of protein wherein the lyophilate comprises at least a protein and any one or a combination of buffer(s), bulking agent(s) and stabilizer(s) and the reconstitution solution comprises salt(s) and optionally buffer(s), bulking agent(s), solvent(s), preservative(s) or a combination thereof.
  • a stable lyophilized dosage form of protein comprises a lyophilate with salt at a concentration of less than or equal to 150 mM and buffer at a concentration of less than or equal to 32 mM respectively.
  • a stable lyophilized dosage form of protein comprises a lyophilate with salt at a concentration of less than or equal to 150 mM along with buffer at a concentration of less than or equal to 32 mM respectively and a reconstitution solution comprising salt(s) and optionally buffer(s), bulking agent(s), solvent(s), preservative (s) or a combination thereof.
  • a stable lyophilized composition of protein comprises a lyophilate with salt at a concentration of less than or equal to 150 mM along with buffer at a concentration of less than or equal to 32 mM respectively and a reconstitution solution comprising salt(s) and optionally buffer(s) and bulking agent(s) or a combination thereof, wherein lyophilized dosage form is qualitatively as well as quantitatively same as that of ready-to-use liquid dosage form of the same protein.
  • the invention relates to a stable lyophilized dosage form of a protein comprising lyophilate which is substantially free of salt.“Substantially free” as used herein, refers to a composition or formulation without additional salts. It is to be understood that the buffer(s) itself may comprise some amount of salt(s).
  • the salt component of a composition refers to a salt apart from the salt present in the aqueous buffer.
  • composition of lyophilized dosage form of protein which is stable at real time and accelerated stress stability conditions for atleast six months and at stress stability conditions for atleast one month.
  • the invention is related to a process for preparation of a stable lyophilized dosage form of protein.
  • a process for producing a stable lyophilized dosage form of protein comprises:
  • Freezing involves equilibrating the vials on the shelf at same temperature ( ⁇ 5°C) and then freezing the solution at defined rates
  • Annealing heating the frozen sample containing protein and buffer system, stabilizer and bulking agent below the collapse temperature.
  • a process for producing a stable lyophilized dosage form of protein comprises the following steps:
  • the invention is related to a kit containing the pharmaceutical dosage form of the invention.
  • the invention is related to a kit containing the pharmaceutical dosage form of the invention wherein the kit comprises two separate containers.
  • the invention is related to a kit comprising a stable lyophilized dosage form of protein wherein a lyophilate with a reduced concentration of salt(s) as well as buffer(s) and a reconstitution solution comprising salt(s) and optionally buffer(s) and bulking agent(s) or a combination thereof is provided, wherein the lyophilized dosage form upon re -constitution is qualitatively as well as quantitatively same as that of ready-to-use liquid dosage form of the same protein.
  • the protein is etanercept.
  • Figure 1 Representative lyophilization cycle used for evaluation of different formulations for cake formation.
  • Figure 2 Lyophilization of ready-to-use liquid formulation of etanercept.
  • Figure 4 Cake appearance for formulations with higher excipient concentration and their characteristics.
  • Figure 5 Cake appearance for different formulations and their characteristics.
  • Figure 6 Stability of formulations 12, 13, 14, 15 and 16 at 40°C after 2 weeks.
  • Figure 7 Stability of formulation 16 at real time stability conditions 2°C to 8°C upto 6 months by SE-HPLC. (a) % Monomer Content, (b) % HMW Content, (c) % LMW Content.
  • Figure 8 (a to c): Stability of formulation 16 at accelerated conditions 25°C/60% RH upto 6 months by SE-HPLC. a) % Monomer Content, (b) % HMW Content, (c) % LMW Content
  • Figure 9 (a to c): Stability of formulation 16 at stress stability conditions 40°C/75% RH upto 1 month by SE-HPLC. (a) % Monomer Content, (b) % HMW Content, (C) % LMW Content.
  • Figure 10 (a to c): Stability of formulation 16 at stress conditions 40°C/75% RH upto 1 month by HI-HPLC. (a) % Main form (b) % Misfolded form (C) % Degraded form. Detail Description of Invention
  • dosage form comprises various pharmaceutically acceptable dosage forms including oral solid as well as liquid dosage forms, such as but not limited to, tablets, soft gelatin capsule, capsules (filled with powders, powders for reconstitution, pellets, beads, mini-tablets, pills, micro-pellets, small tablet units, MUPS, disintegrating tablets, dispersible tablets, granules, sprinkles microspheres and multiparticulates), sachets (filled with powders, pellets, beads, mini-tablets, pills, micropellets, small tablet units, MUPS, disintegrating tablets, dispersible tablets, granules, sprinkles microspheres and multiparticulates) and sprinkles, liquids, liquid dispersions, suspensions, solutions, emulsions, sprays, spot-on) and the like; parenteral dosage forms such as liquids, liquid dispersions, suspensions, solutions, emulsions, (rods, capsules, rings), lyophilized formulation for reconstitution or freeze-dried powder
  • compositions may refer to formulation(s) comprising a protein or a combination of proteins and pharmaceutically acceptable excipient(s) and concentration thereof, prepared such that it is suitable for injection and/or administration into an individual in need thereof.
  • a “composition” may also be referred to as a "pharmaceutical composition” or“formulation” or“pharmaceutical formulation”.
  • the compositions provided herein are substantially sterile and do not contain any agents that are unduly toxic or infectious to the recipient.
  • ready-to-use liquid formulation/dosage form/composition or already established or developed ready-to-use liquid formulation/dosage form/composition refers to a liquid formulation that is supplied in a form which is ready for administration to the patient and is well known to a person skilled in the art. Lyophilate and reconstitution liquid or liquid for reconstitution or solution for reconstitution are terms well known to a person skilled in the art.
  • Reconstituted Solution or solution upon reconstitution as used herein refers to the solution obtained after reconstitution of lyophilate with solution for reconstitution which is ready for administration.
  • Qualitatively as well as quantitatively same as used herein are terms well known to a person skilled in the art.
  • Annealing as used herein means a heat treatment that alters the microstructure of a material and changes its properties, typically by providing defect free crystals and minimizing internal stresses. Annealing typically involves heating to a temperature at which the material is too hard to deform but is soft enough for internal stresses to ease, and then holding the material at that temperature until it is thoroughly equilibrated. In the context of the present invention, annealing particularly involves holding the lyophilate at an annealing temperature for a defined period to ensure crystallization (relatively free of crystal defects) of the crystallizable components in the formulation, particularly the bulking agent. Annealing ensures that such components, particularly the bulking agent, will be stable and completely crystallize the bulking agent during freezing stage.
  • the target product temperature is generally 2 to 5°C below the Tc or Tg’ to get an elegant cake and a short drying cycle.
  • the critical formulation temperature is the eutectic temperature (T eut ) or collapse temperature (T c ), which is commonly determined nowadays by Differential Scanning Calorimetry (DSC). Eutectic temperatures are high relative to glass transitions and processing of such formulations is easy. For amorphous systems, however, the critical temperature is represented by the glass transition temperature (T g ’) of the maximally freeze concentrated solute.
  • T g ’ and T c do not represent the same experimental parameter: T g ’ by DSC is a measure of a heat flow difference between an empty reference pan and a sample pan containing a completely frozen structure. Ice is in close contact with the glassy phase during the entire experiment and is not removed by sublimation (atmospheric pressure). In turn, a Freeze- Dry Microscopy measurement imitates the freeze-drying process by applying controlled, low pressure conditions in a special freeze-drying stage which then allows to sublime ice from the sample. The difference between these two methods becomes obvious in the results obtained for the critical formulation temperature: T c values are many times reported higher (2-5 °C) than their corresponding T g ’ values.
  • Glass transition temperature is the temperature below which a substance loses its elastic properties and becomes hard and brittle (solid) and above which it is elastic (generally, near the transition temperature of glass).
  • “Fill volume” is the volume of dosage form filled in the primary container closure in order to achieve the target deliverable volume.
  • the fill volume accounts to the total volume considering hold up volume and losses during administration.
  • the fluid remaining inside the primary container closure is typically referred to as hold-up volume.
  • “Deliverable volume” is the volume of dosage form that is declared on the label of the drug product.
  • Ionic strength of a solution is a measure of electrolyte concentration and valency of electrolyte.
  • Formulated bulk solution is solution containing the required active pharmaceutical ingredient formulated along with excipients or is diluted to a required concentration prior to lyophilization to produce the dosage form.
  • the term “about” may mean that there can be variation in the concentration of a component of the described compositions that can be to 5%, 10%, 15% or up to and including 20% of the given value.
  • “Treat,” “treating,” or “treatment” are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy.
  • the dosage form comprises fusion protein(s), peptide(s), peptibody, an antibody(s), or an antigen-binding fragment thereof.
  • fusion protein(s), peptide(s), peptibody and antibody(s) are defined in W02007014073 and is incorporated by reference herein.
  • fusion protein(s) comprises a protein fused to an Fc domain that is an antibody constant region, in which the protein means an amino acid polymer made up of several amino acids formed by peptide bonds. Also, a polypeptide has the same meaning as an oligopeptide having a low molecular weight and a protein having a high molecular weight. Thus, as used herein,“a fusion protein of a protein and an Fc domain” has the same meaning as“a fusion protein of a polypeptide and an Fc domain”.
  • the active pharmaceutical ingredient etanercept is used which is obtained from recombinant DNA technology using CHO cells.
  • the concentration of the etanercept in the composition is 10 mg/mL to 100 mg/ml. In a preferred embodiment of the invention, the concentration of etanercept in the composition is 10 mg/mL to 60 mg/ml. In the most preferred embodiment of the invention, the concentration of etanercept in the composition is 20 mg/mL to 60 mg/ml.
  • “High concentration" as used herein with reference to protein therapeutic compositions means 20 mg/ml or more.
  • Tumor Necrosis Factor alpha is a member of a group of cytokines that stimulate the acute phase reaction, and thus is a cytokine involved in systemic inflammation. TNF-alpha is able to induce inflammation, induce apoptotic cell death, and to inhibit tumorgenesis and viral replication.
  • Dysregulation of TNF-alpha production has been implicated in a variety of human diseases like autoimmune disease, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, Wegener's disease (granulomatosis), Crohn's disease or inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, atopic dermatitis, Alzheimer as well as cancer.
  • autoimmune disease ankylosing spondylitis
  • juvenile rheumatoid arthritis psoriasis
  • psoriatic arthritis psoriatic arthritis
  • rheumatoid arthritis Wegener's disease (granulomatosis), Crohn's disease or inflammatory bowel disease
  • COPD chronic obstructive pulmonary disease
  • Dosage of the etanercept will depend on the disease, severity of condition, patient's clinical history, and response to the (prior) therapy, and will be adjusted and monitored by a physician.
  • the pharmaceutical composition may be administered parenterally, such as subcutaneously, intramuscularly, intravenously, intraperitoneally, intra-cerebrospinally, intra-articularly, intra-synovially and/or intra-thecally by either bolus injection or continuous infusion.
  • etanercept may be administered in adult or juvenile subject, wherein the amount may range from about 1 - 80 mg.
  • the dose may be administered once weekly, twice weekly. Further, the doses may be administered weekly, biweekly, or separated by several weeks e.g. three weeks.
  • the therapeutic dose and duration may vary as per patient response and patient requirement.
  • a suitable regimen for juvenile and pediatric patients may involve a dose of 0.4 mg/kg to 5 mg/kg of etanercept, administered one or more times per week. In case of adult rheumatoid arthritis, 25 mg twice weekly or 50 mg once weekly etanercept may be administered.
  • 25 mg twice weekly or 50 mg once weekly etanercept may be administered.
  • 25 mg twice weekly or 50 mg once weekly etanercept may be administered.
  • the recommended dose of etanercept is 25 mg administered twice weekly or 50 mg administered once weekly.
  • the recommended dose of etanercept is 0.8 mg/Kg weekly with a maximum of 50 mg dose per week.
  • the recommended dose of etanercept is 0.8 mg/Kg weekly with a maximum of 50 mg dose per week.
  • compositions described herein are prepared in a bulk formulation and as such, the components of the composition are adjusted so that it is higher than would be required for administration and diluted appropriately prior to administration.
  • the compositions described herein are administered parenterally, e.g., subcutaneously, intramuscularly, intravenously, intraperitoneal, intracerebrospinal, intraarticular, intrasynovial, and/or intrathecal.
  • long-term storage is understood to mean that the composition can be stored for three months or more, for six months or more, or for one year, or two years, or more. Long term storage is also understood to mean that the active polypeptide of the pharmaceutical composition does not substantially lose its activity as compared to the composition at the beginning of storage.
  • stable with respect to long-term storage refers to the appearance (elegance, compactness, brittleness) of the cake formed by incorporating the compositions and the process for preparing the composition of the invention. Stability of a composition can be assessed based on purity by SE-HPLC (Size exclusion Chromatography) and HI-HPLC (Hydrophobic interaction chromatography).
  • compositions described herein are presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the invention is related to a kit or container containing the pharmaceutical composition of the invention.
  • Lyophilized compositions in various aspects and embodiments of the invention may be reconstituted by a variety of methods, including, for instance, the procedures specified by a manufacturer and/or supplier for reconstituting a pharmaceutical protein comprising lyophilate composition for veterinary or human use.
  • Reconstitution Time For timing reconstitution, a stop watch should be started as soon as the diluent is started into the vial. After the desired volume of diluent has been introduced, the vial is swirled gently for preferably 10 seconds. Reconstitution should be monitored under controlled conditions with a known intensity of light incident on the sample, against uniform black and uniform white backgrounds.
  • the lyophilate has reduced concentration of salt.
  • Reduced concentration of salt means salt at a concentration of less than or equal to 150 mM, more preferably less than or equal to 30mM.
  • lyophilate is substantially free of salt.“Substantially free” as used herein, refers to a composition or formulation without additional salts. It is to be understood that the buffer(s) itself may comprise some amount of salt(s).
  • the salt component of a composition refers to a salt apart from the salt present in the aqueous buffer.
  • Salts used herein, can include without limitation sodium chloride, potassium chloride, magnesium sulphate, calcium chloride, magnesium chloride, sodium nitrate, sodium chromate, magnesium dioxide, potassium nitrate, sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, sodium acetate, potassium acetate, calcium acetate or magnesium acetate.
  • the salt is sodium chloride (NaCl).
  • D. Buffer(s) is sodium chloride (NaCl).
  • Buffer(s) in accordance with this aspect of the invention are compatible with the protein appropriate to the desired end use, provide adequate buffering capacity at concentrations consistent with acceptable osmolality, are inert, stable, and have their maximum buffering capacity at or near the desired pH.
  • a variety of buffers can be used in the lyophilization solution in accordance with various aspects and preferred embodiments of the invention in this regard. (Some of the same buffers can be used to reconstitute lyophilate, as discussed below.)
  • Preferred buffers in this aspect of the invention include Tris, phosphate, citrate and citrate-phosphate, acetate, succinate to mention just a few.
  • the buffer concentration is less than or equal to 32 mM. In another embodiment the buffer concentration is in the range of 5 to 50 mM. Buffers in accordance with this aspect of the invention are effective to maintain appropriate pH. The exact optimal pH will vary from protein to protein. Accordingly, different buffer systems will be more or less better than one another for different proteins. Generally, however, the preferred buffers are effective for pH in the range of 5 to 8, especially in the range of 5.5 to 7.5. E. Bulking Agents
  • bulking agents can be used in accordance with various aspects and preferred embodiments of the invention herein disclosed.
  • bulking agents can be used in this regard to facilitate the formation of uniform lyophilate cakes with desired structure and porosity.
  • bulking agents useful in this regard are mannitol, anhydrous lactose, sucrose, D(+)- a,a-trehalose, dextran 40, povidone (PVP K24), glycine, and hydroxyethyl starch.
  • glycine a particularly preferred bulking agents in this is glycine.
  • Lyoprotectants/Stabilizers are substances that, generally, protect proteins against denaturation as a result of lyophilization.
  • lyoprotectants are used in the lyophilization process, and the resulting lyophilates and compositions produced thereby comprise the same.
  • lyoprotectants in accordance with certain preferred embodiments of the invention is sucrose.
  • the lyophilized formulation according to the present invention may further include a preservative.
  • the preservative may include benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chloro-cresol, phenylmercuric nitrate, thimerosal, and benzoic acid, but are not limited thereto.
  • These preservatives may be used either alone or in combinations of two or more thereof.
  • any one of the compositions described herein can comprise an excipient.
  • the excipient may be sucrose, lactose, glycerol, xylitol, sorbitol, Mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin (BSA), human SA or recombinant HA, dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethylene glycol, ethylene glycol, glycerol, dimethysulfoxide (DMSO), dimethylformamide (DMF), proline, L-serine, sodium glutamic acid, alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyric acid, Tween-20, Tween-80
  • Freezing involves equilibrating the vials on the shelf at same temperature ( ⁇ 5°C) and then freezing the solution at defined rates
  • Annealing heating the frozen sample containing protein and buffer system, stabilizer and bulking agent below the collapse temperature.
  • Enbrel ® is a marketed formulation of etanercept for subcutaneous injection, supplied as ready-to-use liquid formulation in prefilled syringe, or as prefilled syringe in an auto injector, or as a lyophilized product in a glass vial which is reconstituted with solution for reconstitution before injection.
  • the composition of Enbrel ® ready-to-use liquid and lyophilized dosage form is as follows: Table 1(a) Composition of Enbrel ® ready-to-use liquid formulation.
  • Example 1 Lyophilization of etanercept ready-to-use liquid formulation.
  • Table 2 Composition of etanercept ready-to-use liquid formulation (Control DS) and role of excipients
  • Table 2 enlists the excipients and their role in ready-to-use liquid formulation for etanercept.
  • Glycine and sucrose present in ready-to-use liquid formulation could act as bulking agent and support cake formation.
  • the ready-to-use liquid formulation as such was lyophilized using lyophilisation cycle (Figure 1) to test the cake formation and its attributes. Surprisingly, no visible cake was observed and the dried material resembled to that of liquid drying ( Figure 2). It is possible that one or more of formulation excipients could be interfering with cake formation. This can happen by excipients modulating physical properties of bulk solution pertaining to freezing and/or drying process. Additionally it could also be possible that cake formation required a critical concentration of bulking agent/s.
  • Table 3 Compositions evaluated for cake formation Bulking agents affected the elegancy of cake in both buffer systems ( Figure 3). Glycine as a bulking agent resulted in lustrous cake formation with cake volume equivalent to fill volume. On the other hand partial collapse of cake was observed when trehalose and sucrose were used as bulking agent. However, both, sucrose and trehalose have been historically used as bulking agents, and it is possible that by modifying the lyophilization cycle, a cake with desired attributes can be obtained.
  • Example 3 Use of higher concentration of excipients to obtain cake upon lyophilization.
  • ready-to-use liquid formulation consisted of 50 mg/ml etanercept and the fill volume was 1.0 ml.
  • etanercept 50 mg/ml with two-fold (2x) concentration of bulking agents and etanercept 75 mg/ml with three-fold (3x) bulking agent concentration were lyophilized.
  • Table 4 describes various compositions and their respective reconstitution buffers).
  • NaCl was excluded along with reduction of buffer strength from concentrated formulations as presence of higher salt content reduces the Tg’ and results in collapse of the cake.
  • Example 4 Optimization of formulation compositions for lyophilisation and reconstitution buffer for desirable cake attributes which is qualitatively and quantitatively same as that of ready-to-use liquid formulation
  • excipients interfering the cake formation higher sodium chloride content and higher buffer strength
  • the formulations tried with this approach are as follows:
  • Table 5 Composition obtained by removing or reducing the interfering excipients and adding it to solution for reconstitution.
  • the solution for reconstitution may or may not contain 0.9% benzyl alcohol as preservative.
  • the formulations form the final cycle (Formulation number 13, 14, 15 and 16) were charged on stability at 40°C for 2 weeks to evaluate further shrinkage or melt back due to moisture content.
  • Formulation No. 12 Cracked cake Formulation No. 13 : Elegant cake but with shrinkage Formulation No. 14 : Elegant cake but with shrinkage Formulation No. 15 : Elegant lustrous cake with minor shrinkage Formulation No. 16 : Elegant lustrous cake with minor shrinkage
  • the reconstitution time of all the formulation was less than 2 minutes.
  • formulation no. 13, 14, 15 and 16 formed a stable cake and had no major changes.
  • the cake of formulation no. 15 and 16 was comparable to the marketed formulation (Enbrel®).
  • the formulation no. 12 formed an elegant but brittle cake which cracked during handling.
  • the moisture content of formulation no. 15 and 16 was determined by reconstituting the Hydranal solution in the etanercept lyophilized drug product vial followed by centrifugation and injecting the supernatant in Karl fisher for moisture content estimation.
  • the moisture content values were found to be ⁇ 2% w/w.
  • the data for moisture content is as given below.
  • Table 7 Moisture content of formulation no. 15 and 16 in lyophilization cycle trial 14, 15 and 16.
  • the lyophilization cycles used for development of these formulations involved freezing of drug substance from -40°C to -50°C at a rate of ⁇ 0.6°C/min to l .5°C/min followed by and annealing at -25°C to -30°C at a rate of 0. l°C/min to 0.3°C/min.
  • Slow freezing results in formation of larger ice crystals and interstitial pores and in turn increases mass/moisture transfer during drying process.
  • Faster rate of drying forms smaller ice crystals and interstitial pores in turn reducing the rate of moisture transfer during drying process and increasing moisture content in the lyophilized cake.
  • Primary and secondary drying involved stepwise increment of temperatures and holding at each step for 4 to 6 hours.
  • the pressure in the lyophilization chamber was maintained at 100 to 50 mTorr during primary and secondary drying.
  • the total lyophilization cycle varied from 48 to 72 hours during development stage. The final cycle was approximately of 48 hours.
  • Example 6 Comparison between formulated bulk solution of formulation 15 and 16: The formulated bulk for formulation 15 and 16 were analyzed for its purity after buffer exchange step and table 8 below summarizes the results obtained from SE-HPLC analysis.
  • Table 8 Comparison between purity of formulated bulk for formulation 15 and 16 by SE- HPLC.
  • Example 7 Comparison of Tm (melting point) values of formulation 15 and 16 by DSC (Differential Scanning Colorimetry)
  • Tmi, Tm 2, and Th3 ⁇ 4 for etanercept Three thermal transition temperatures of Tmi, Tm 2, and Th3 ⁇ 4 for etanercept were observed. These indicated unfolding states at an extra-cellular ligand binding portion, tumour necrosis factor receptor (TNFR; Tml) and the presence of two domains originating from the Fc component of Etanercept containing the domains of CH2 (Tm2), CH3 (Tm3).
  • TNFR tumour necrosis factor receptor
  • Tm3 tumour necrosis factor receptor
  • T m values of formulation number 15 and formulation number 16 are summarized in table 9 below.
  • Table 9 Melting temperatures (Tm) of formulation 15 and 16 by DSC.
  • Tm values of formulation number 15 are significantly lower than the Control DS.
  • formulation 16 had T m values higher than formulation 15 and were close to the Control DS, suggesting improved stability.
  • Tml representing unfolding of the TNFR receptor and Tm2 representing unfolding CH2 domain are considered key indicators of stability by nano-DSC analysis.
  • Formulation 16 was prepared and lyophilized according to lyophilization cycle described in Example 5.
  • the lyophilized formulation was incubated at real time (2°C to 8°C), accelerated (25 °C) and stressed conditions (40°C).
  • the lyophilized formulation at different time point i.e. after 1 month for stressed conditions and after 6 months for real time and accelerated conditions
  • Figure 10 summarizes the stability data by HI HPLC for formulation 16 at stressed conditions 40°C/75% RH upto 1 month.
  • formulation number 16 did not have any impact on the purity by HI HPLC upto one month’s time.
  • Table 11 pH formulation 16 at real time conditions (2°C to 8°C) upto 3 months.
  • the formulations of the invention have several benefits relative to other protein lyophilized composition, including reduced cost of development, minimum reconstitution time, minimum particulates upon reconstitution and a relatively stable elegant commercial viable protein lyophilized formulation.
  • a ready-to-use liquid formulation which does not form cake as such can be modified as described here resulting in elegant cake.
  • This study thus exemplifies lyophilization strategy to convert an established or developed ready-to-use solution liquid dosage form of protein to lyophilized dosage form of the same protein which upon reconstitution has the same composition as that of the ready-to-use liquid formulation.

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EP19759046.6A 2018-06-26 2019-06-26 Stabile lyophilisierte darreichungsform von protein Withdrawn EP3813791A2 (de)

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